The growth potential of mammalian embryonic stage cells have been known for many years, but the ability to culture such pluripotent and totipotent stem cells, particularly human stem cells, has only been recently developed. Stem cells have a capacity both for self-renewal and the generation of differentiated cell types. Embryonic stem (ES) cells are derived from cultures of inner cell mass (ICM) cells, and have the property of participating as totipotent cells when placed into host blastocysts. The developmental pathways that endogenous ICM cells or transferred ES cells take to tissue formation and organogenesis has led many to hope that these pathways can be controlled for the development of tissue and organ specific stem cells.
Human ES cells have a variety of research and potential clinical uses. Diseases that might be treated by transplanting human ES-derived cells include Parkinson's disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's muscular dystrophy, heart failure, and osteogenesis imperfecta. One of the advantages of using ES cells as compared to adult stem cells is that ES cells have an unlimited ability to proliferate in vitro, and are more likely to be able to generate a broad range of cell types through directed differentiation.
Human ES cells can also be used to study early events in human development. Still-unexplained events in early human development can result in congenital birth defects and placental abnormalities that lead to spontaneous abortion. By studying human ES cells in vitro, it may be possible to identify the genetic, molecular, and cellular events that lead to these problems and identify methods for preventing them. Such cells could also be used to explore the effects of chromosomal abnormalities in early development. This might include the ability to monitor the development of early childhood tumors, many of which are embryonic in origin.
Human ES cells can also be used to test candidate therapeutic drugs. Currently, before candidate drugs are tested in human volunteers, they are subjected to a barrage of preclinical tests. These include drug screening in animal models—in vitro tests using cells derived from mice or rats, for example, or in vivo tests that involve giving the drug to an animal to assess its safety. Although animal model testing is a mainstay of pharmaceutical research, it cannot always predict the effects that a candidate drug may have on human cells. For this reason, cultures of human cells are often employed in preclinical tests. These human cell lines have usually been maintained in vitro for long periods and as such often have different characteristics than do in vivo cells. These differences can make it difficult to predict the action of a drug in vivo based on the response of human cell lines in vitro.
Human ES cells can also be employed to screen potential toxins. The reasons for using human ES cells to screen potential toxins closely resemble those for using human ES-derived cells to test drugs (above). Toxins often have different effects on different animal species, which makes it critical to have the best possible in vitro models for evaluating their effects on human cells.
Human and mouse ES cells differ in morphology, immunophenotype, and growth properties. While mouse ES cells grow in attached rounded masses in which single cells are difficult to identify, the primate cells grow in flat colonies with varying distinctness of cell borders in monolayer culture. Like mouse ES and embryonic germ (EG) cells, primate pluripotent cells, including some human EC cells, require a mouse embryonic fibroblast feeder-cell layer for support. In the case of mouse ES and EG cells, the feeder cell requirement can be replaced by LIF or related members of this cytokine family, but pluripotent human EC cells, rhesus monkey ES cells, and human ES cells will not respond to LIF in such a fashion. Even on a feeder cell layer, all primate pluripotent cells grow very poorly when dissociated to single cells, whereas mouse ES cell lines can be cloned at a relatively high efficiency in the presence of LIF under these conditions.
For many purposes, it is desirable to have the ability to store human or other primate ES cells for long periods of time. Mouse ES cells are conveniently frozen, or cryopreserved, for such purposes. However, such methods have not proven to provide for viable cryopreservation of primate ES cells.
There are previously reported techniques for hESC cryopreservation. Reubinoff et al., 2001, Human Reprod. 16, 2187 describes a vitrification method, and Ji et al., 2004, Biotechnol. Bioeng. 88, 299 describes freezing cells in adherent culture within a matrigel matrix. The vitrification technique is technically challenging. High levels of cryoprotectants are toxic at room temperature requiring strict attention to time and temperature constraints during freeze and thaw, cell growth following thaw is not robust and the freezing container has such a large surface to volume ratio that the cells are sensitive to routine handling while frozen. The matrix embedding method improves survival over standard freezing. However, because hESC are frozen adherent to the culture dish, liquid nitrogen storage becomes problematic since most liquid nitrogen storage vessels are not designed to hold culture plates. They can be conveniently stored at −80° C. But, −80° C. storage substantially reduces the time cells can maintain viability while frozen. In addition, freezing in a matrix must be anticipated by at least 24 hours.
Methods that provide for stable storage and high viability of primate ES cells are of great interest for many purposes. The present invention addresses these needs.
Publications
Mazur (1977), “Slow freezing injury in mammalian cells”, p. 19-42. In K. Elliott and J. Whelan (Eds.), The Freezing of Mammalian Embryos, Ciba Found. Symp. 52, Elsevier Excerpta Medica, Amsterdam. Leibo and Mazur (1978) “Methods of the preservation of mammalian embryos by freezing”, p. 179-201. In J. C. Daniel Jr., (Ed.) Methods of Mammalian Reproduction, Academic Press, New York, N.Y. Leibo (1981) “Preservation of ova and embryos by freezing”, p. 127-139. In B. G. Brackett, G. E. Seidel Jr., S. M. Seidel (Eds.) New Technologies in Animal Breeding. Academic Press, New York, N.Y. Heng et al. (2004) The Cryopreservation of Human Embryonic Stem Cells” Biotechnol. & Appl. Biochem. Kim et al. (2004) Stem Cells 22:950-961, “Effects of Type IV Collagen and Laminin on the Cryopreservation of Human Embryonic Stem Cells.” Fujioka et al. (2004) Int. J. Dev. Biol. 48:1149-1154, “A Simple and Efficient Cryopreservation Method for Primate Embryonic Stem Cells.”